Gene delivery systems can be broadly classified into two groups: viral and nonviral. Viral systems have major toxicity risks and have resulted in major complications and death in clinical trials. Nonviral systems are far less efficient than viral approaches but offer the potential to tailor applications to enhance specificity and potentially decrease toxicity. Nonviral strategies can be broadly classified as lipid-based or nonlipid-based. The strategy presented in this invention can be applied to any of the existing nonviral approaches, so all will be described here.
The simplest nonviral system is direct delivery of DNA. Due to the negative charge of DNA, very little of the DNA actually enters the cell and most is degraded. Virtually none of the DNA enters the nucleus without a nuclear targeting sequence in the strategy. Conventionally, another factor is employed to enhance the efficiency of gene/product delivery (DNA, RNA, or more recently protein therapeutics) either by mechanical effects such as electroporation, ultrasound, “gene gun” and direct microinjection, or by charge neutralization and chemical effects with agents such as calcium phosphate, polylysine, and liposome preparations. In the latter strategies, charge neutralization has been shown to increase nonspecific efficiencies several-fold over even chemical/mechanical effects of liposome preparations alone. Based upon these and similar results, many have concluded that DNA and RNA require charge neutralization for efficiency in cellular uptake, since DNA's negative charge essentially precludes transport except by endolysis with subsequent lysosome fusion (escaped with addition of other agents). Most transfection agents actually use an excess of positive charge in ratios of 2-4 fold over the net DNA negative charge. The resulting positive hybrid binds ionically to negatively-charged cell surface proteoglycans and dramatically enhances subsequent uptake. Some transfection agents seem to have a cellular tropism, most likely because of steric and charge patterns that more effectively target particular proteoglycans, which vary in cell-type specific patterns. Even with appropriate agents (i.e., correct tropism), charge neutralization alone or in combination with liposomes remains extremely inefficient relative to viral strategies. Thus, the community has identified a number of peptides and peptide fragments which facilitate efficient entry of a complex into a cell and past any endolysosome stage. Several such transport factors even allow efficient nuclear entry. In one process, the transport factor is directly linked to the therapeutic product of interest (small drug, gene, protein, etc). This approach requires that a new drug attached to the transport factor be produced, purified and tested. In many cases, these hybrids will actually constitute new drugs and will require full testing. Such a process results in significant additional risk and expense. Alternately, a number of strategies merely employ mixing of the agent nonspecifically (or even specifically at the surface) into liposome preparations as carriers for a drug/DNA/factor. Although an improvement over direct or simpler modalities in terms of efficiencies, these approaches remain inefficient (relative to virus) and considerably more toxic than simple nonviral strategies. Part of this inefficiency is due to poor nuclear translocation. As a result, strategies have evolved to add nuclear translocation signals to the complex detailed above, either as part of the therapeutic factor hybrid or as part of the liposome mixture. Additional refinements have included efforts to reduce DNA/RNA/factor degradation.
Perhaps the most important refinements in the basic strategies presented above have included specific ligands or other targeting agents together with the therapeutic factor. These strategies offer the potential for greatly reduced nonspecific toxicity and substantial improvements in efficiency, particularly when combined with efficiency agents described as above. However, the current strategies rely on covalent linkages to a single carrier and thus necessitate a specific synthesis (to assure that steric considerations in a degree of substitution scheme don't favor a single factor over the others—i.e., to assure that each efficiency factor and each imaging moiety, and each targeting moiety is present on the backbone). This renders virtually impossible a number of specific constructs (for example, sialyl-lewis X and an Fab fragment to a surface antigen, since steric limitations would prevent efficient binding of one or the other in most schemes, and in turn would interfere with efficiency factors). While promising in concept, these approaches represent expensive, very low yield (in terms of synthesis), and unproven solutions to this problem.
As must be evident, with each stage of development in nonviral gene and factor delivery, problems have been encountered and, in the next stage, solved with an added degree of complexity. Each improvement represented an incremental step over the prior standard. However, the added complexity brings risk from a patient-care standpoint and inefficiency and expense from a production standpoint. These barriers have led to greatly decreased enthusiasm for these otherwise promising potential therapies.
What is needed are new methods and compositions that are broadly applicable to compositions of diverse therapeutic or cosmoceutic agents, that can be targeted or imaged to maximize delivery to a particular site. Surprisingly, the present invention provides such compositions and methods.